Methanosphaera Medium 1

[所属分类:培养基配方] [发布时间:2021-5-26] [发布人:网站管理员2] [阅读次数:] [返回]

Methanosphaera Medium 1

山东拓普生物工程有限公司 培养基配方 http://www.topbiol.com

Composition per liter:

KH 2 PO 4 .........................................................................................2.8g
Trypticase™..................................................................................2.0g
Yeast extract..................................................................................2.0g
NH 4 Cl ...........................................................................................1.0g
K 2 HPO 4 .........................................................................................0.6g
NaCl..............................................................................................0.6g
Sodium acetate..............................................................................0.5g
Sodium formate ............................................................................0.5g
(NH 4 ) 2 SO 4 ....................................................................................0.3g
MgSO 4 ·7H 2 O..............................................................................0.15g
CaCl 2 ·2H 2 O ..............................................................................0.076g
FeSO 4 ·7H 2 O...............................................................................3.0mg
Na 2 SeO 4 .....................................................................................1.9mg
Resazurin ...................................................................................1.0mg
NiCl 2 ·6H 2 O................................................................................0.7mg
Rumen fluid, clarified............................................................100.0mL
Trace elements solution...........................................................10.0mL
L -Cysteine·HCl·H 2 O solution..................................................10.0mL
Na 2 S·9H 2 O solution.................................................................10.0mL
Methanol solution......................................................................5.0mL
Vitamin solution.........................................................................1.0mL
pH 6.5 ± 0.2 at 25°C
Trace Elements Solution:
Composition per liter:
MgSO 4 ·7H 2 O............................................................................... 3.0g
Nitrilotriacetic acid.......................................................................1.5g
NaCl..............................................................................................1.0g
MnSO 4 ·2H 2 O............................................................................... 0.5g
CoSO 4 ·7H 2 O...............................................................................0.18g
ZnSO 4 ·7H 2 O...............................................................................0.18g
CaCl 2 ·2H 2 O...................................................................................0.1g
FeSO 4 ·7H 2 O..................................................................................0.1g
NiCl 2 ·6H 2 O...............................................................................0.025g
KAl(SO 4 ) 2 ·12H 2 O.......................................................................0.02g
CuSO 4 ·5H 2 O...............................................................................0.01g
H 3 BO 3 .........................................................................................0.01g
Na 2 MoO 4 ·2H 2 O ......................................................................... 0.01g
Na 2 SeO 3 ·5H 2 O...........................................................................0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Adjust pH to 7.0. Add distilled/de-
ionized water to 1.0L.
L -Cysteine·HCl·H 2 O Solution:
Composition per 10.0mL:
L -Cysteine·HCl·H 2 O .................................................................0.875g
Preparation of  L -Cysteine·HCl·H 2 O Solution: Add  L -cystei-
ne·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL.
Mix thoroughly. Autoclave under 80% N 2 + 20% CO 2 for 15 min at 15
psi pressure–121°C.
Na 2 S·9H 2 O Solution:
Composition per 10.0mL:
Na 2 S·9H 2 O................................................................................0.375g
Preparation of Na 2 S·9H 2 O Solution:  Add Na 2 S·9H 2 O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pres-
sure–121°C.
Methanol Solution:
Composition per 10.0mL:
Methanol..................................................................................10.0mL
Preparation of Methanol Solution:  Sparge 10.0mL of methanol
with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C.
Vitamin Solution:
Composition per 10.0mL:
Calcium- D -pantothenate ..........................................................20.0mg
Nicotinamide............................................................................20.0mg
Pyridoxine·HCl........................................................................20.0mg
Riboflavin ................................................................................20.0mg
Thiamine·HCl ..........................................................................20.0mg
Biotin .......................................................................................10.0mg
p-Aminobenzoic acid.................................................................1.0mg
Folic acid....................................................................................0.5mg
Vitamin B 12 ................................................................................0.2mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Filter ster-
ilize. Sparge with 80% N 2 + 20% CO 2 .
Preparation of Medium: Add components, except methanol solu-
tion,  L -cysteine·HCl·H 2 O solution, and Na 2 S·9H 2 O solution, to dis-
tilled/deionized water and bring volume to 975.0mL. Mix thoroughly.
Gently heat and bring to boiling. Continue boiling for 5 min. Cool to
room temperature while sparging with 100% N 2 . Autoclave for 15 min
at 15 psi pressure–121°C. Aseptically and anaerobically add 10.0mL of
sterile  L -cysteine·HCl·H 2 O solution, 10.0mL of sterile Na 2 S·9H 2 O so-
lution, and 5.0mL of sterile methanol solution. Mix thoroughly. Asep-
tically and anaerobically distribute into sterile screw-cappped bottles
under 100% N 2 .
Use: For the cultivation and maintenance of Methanosphaera stadt-
manae.